S. H. Hutner

NUTRITION OF SOME PHAGOTROPHIC FRESH-WATER CHRYSOMONADS

The chrysomonads are a vast array of brown-pigmented fresh-water, soil, and marine flagellates. Colorless phagotrophic counterparts of several of the pigmented species are common in nature, e.g., Molzus and Oikomonus. The minimal nutritional requirements of 4 fresh-water strains belonging to 2 species are described here. No chrysomonad previously has been grown in chemically defined media. These strains ingest particulate food and are therefore animals (protozoa) and, being to some extent phototrophic, they are plants (algae) as well. They can live indefinitely in darkness or in light on high-molecular dissolved food, as well as in low-molecular chemically defined media. This extraordinary versatility, first thoroughly documented in the pioneering pure culture study of Pring~heirn,~ is accompanied by a remarkable tolerance of variations in media and exceedingly dense growth in appropriate media, including certain simple “synthetic” media [one cannot unambiguously call “synthetic” a medium containing cobalamin (vitamin Biz), whose complete structure is still to be ascertained]. This ease of cultivation-in which respect these chrysomonads contrast sharply with some of the planktonic chrysomonadslO-suggests that the biochemical basis of an intermeshed phagotrophy and phototrophy can profitably be investigated in them. PringsheimQ reviewed the scanty previous work. He demonstrated an active phagotrophy in the same strains on which the present paper is based: They ingested starch grains, oil droplets, casein particles, bacteria, and small algae -including each other. Glucose was utilized in the presence of complex natural materials such as peptone. Liver extracts were strikingly stimulatory. Dark growth was improved by aeration and paralleled that in light. Darkgrown individuals were pale. Cultures accumulated a water-soluble sepia or almost black substance which was also produced by pale growths in the dark. With chemically defined media-the subject of this paper-it should be possible to explore systematically their ability to utilize high-molecular sources of energy and carbon, nitrogen and amino acids, biotin, thiamine, and cobalamin. To identify the growth requirements of aerobic phototrophs, culture methods are needed which combine adequate illumination with freedom from significant chemical contaminations. Such considerations apply with special force to these chrysomonads. Growing densely, they need much illumination. Hence they were cultured in shallow layers of medium, and their sensitive response to certain widely-distributed growth factors such as biotin and cobalamin spurred the development of methods to minimize the confusion introduced by

Nutrition of Herpetomonas (Strigomonas) culicidarum.

No group offers more diverse and numerous species for the study of the comparative biochemical evolution of parasitism than does the Trypanosomidae. Unfortunately, little biochemical study of this family has been possible because of the difficulty encountered in cultivation and maintenance of many ~pecies.2~~ 6 Indeed, among the pathogens some have been impossible to culture ia d r o (e.g., Trypanosoma equiperdum, Trypanosoma congolense) and, in others, where cultures have been obtained, infectivity has not always been retai11ed.2~ In view of the limited success in identifying growth factors for exacting members of the Trypansomidae,16 it was thought that other, nonpathogenic parasites found in this family, and possessing relatively simple growth requirements, would lend themselves more easily to nutritional studies. It was assumed that certain physiological characteristics would be common to both pathogenic and non-pathogenic groups, and that consequently a study of a representative of the latter group would be a stepping-stone to the identification of requirements for the more fastidious groups. The blood requirement of many trypanosomes and Leishmania has been difficult to satisfy with simpler materials. Certain parasites grow in the presence of hemin plus serum as a blood substitute (Leishmania agamae, Leishmania ceramodactyli) but many serum requirements still defy analysis15 ( c j . SprinceZ0). Herpetomonas culicidarum, a mosquito parasite in the family Trypanosomidae, has, however, a blood requirement that can be completely replaced by hemin.16 The remaining growth factors are thermostable and are found in ordinary peptones. These considerations plus the ease of handling and lack of pathogenicity made us suppose that the nutritional requirements of this parasite could be identified by procedures successful with many exacting protists. Its morphological relationship to Leishmania and to Trypanosoma cruzi indicated that its nutritional requirements might be similar in the main to both these pathogenic groups. Herpetomonas culcidarumt was first isolated by Noguchi and Tilden16 in 1926 by streaking a saline suspension of infected mosquito gut on blood agar. M. Lwoff demonstrated in 1940,15 with the aid of hydrolyzed silk, that thiamine was essential. Little work has been done since. Materials. Experimental media were contained in 25-ml. flasks incubated at 25-28OC. The total volume in each flask was 5 ml. The flasks were covered

NUTRITION OF PERANEMA

Animals, designated broadly as phagotrophs, are diverse in origin. Organization of their comparative biochemistry is well under way based largely on data from a few metazoa (mammals, birds, and insects) and ciliates (Tetrahymena and Paramecium). Theories about the biochemical features characterizing animals, if derived exclusively from species remote from their presumably photosynthetic ancestors, are too narrowly founded. Later specializations reflecting a restricted range of prey, or adaptations to cellularity may obscure common features. Certain flagellate groups comprise both phototrophs and phagotrophs and are therefore of special value. Indeed, in certain chrysomonads, phagotrophy and phototrophy co-exist in the same organism. But before generalizing about which components of the photosynthetic apparatus survive in the dark metabolism of phagotrophs, other plantjanimal groups, such as the Euglenineae, should be looked into. In recent years, understanding of the nutrition and intermediary metabolism of phototrophic euglenids has deepened, but the animal euglenids remained untouched. Consequently, when Professor E. G. Pringsheim presented us with a pure culture of the colorless voracious euglenid Peranema trichophorum, it provided the opportunity, for which we are most grateful, to examine the nutrition of a phagotroph belonging to a line rich in (1) photosynthetic species widely divergent in capacity for heterotrophic nutrition; (2) colorless osmotrophs; and (3) phagotrophs. No photosynthetic euglenid is known to be phagotrophic; in this respect, the euglenid series is incomplete as compared with the chrysomonads and dinoflagellates. The use of Euglena gracilis for assaying cobalamin lent a practical incentive to this investigation. Euglena is limited in ability to utilize high-molecular cobalamin-a shortcoming of the assay.’’ 8 , If a cobalamin requirement is phyletic in euglenids, then Peranema should need cobalamin and, as a phagotroph, serve for assaying total cobalamin in high-molecular materials, even in intact cells, without chemical or enzymatic digestion being needed to render the vitamin assayable. Chen’ first cultivated P. trichophorum on living Saccharomyces exiguus + soil extract. Thriving cultures were then obtained on autoclaved cows’ milk. Chen’s observations depict it as strikingly omnivorous and aggressive-in his words: “Peranema is capable of ingesting a great variety of living organisms providing these are motionless. Small organisms are swallowed whole; larger ones are either engulfed or cut open by the rod-organ and their contents sucked out, The rod-organ can be protruded out of the cytostome and used in holding on to, and cutting, the periplast of the prey. Starch-grains, oildroplets, and protein-particles are engulfed and digested.”

Age as a Factor in Susceptibility of Mice to the Endotoxin of Bacillary Dysentery.

Summary Dose-survival curves obtained by injecting the endotoxin of Shigella paradysenteriæ Flexner into 12-, 20-, and 35-g mice indicate that such mice are killed by approximately the same minimal amounts of toxin, and that this effect is not considerably modified within the limits of age or normal weight indicated. This effect is interpreted as injury to a single system, most probably the vascular system.

USE OF AMINE BUFFERS IN PROTOZOAN NUTRITION

The toxicity for a protozoan of certain amine buffers, including tris(hydroxymethy1)aminomethane (Tris), is one phase of an extensive study of the reliability of certain protozoa as guides to the metabolism of higher animals. This paper describes the remarkable low toxicity of Tris towards our main test organism, the phagotrophic (particle-ingesting), brown-pigmented photosynthetic flagellate Ochromonas danica. The culture medium used was devised to permit growth at a pH (7.6) a t least as high as that of blood. Since this flagellate has exceptionally high metal requirements, supplying sufficient iron and trace elements in an alkaline medium seemed comparable to the problem of securing sufficient absorption of iron and other trace elements by an achlorhydric athlete. Media of the type to be described may prove useful for detecting basic cytotoxic materials. Soon after their introduction to biology as buffers for histochemical work, Tris and related buffers came into general use in microbiology. A precaution in its use in mammalian systems was implied by the findings of Macleod and Onofrey (1954) : Tris and triethanolamine competitively interfered with the uptake of K+ by Lactobacillus arabinosus. The advisability of fortifying with K+, Tris-buff ered media for marine algae and phytoflagellates was emphasized by Provasoli et al. (1957), who noted that at fiH 7.5 and 8.5, 100 mg. per cent Tris raised the minimal requirement of the blue-green alga Phormidium persicinum from 0.5 mg. per cent (the minimal concentration tried) to 4 to 5 mg. per cent. Similar precautions were advised for freshwater algae (Provasoli and Pintner, 1960), but with some variations: the toxicity of triethanolamine was counteracted by increasing the Ca concentration for Volvox globator and by Ca + Mg for V . tertius and a peridinian. The toxicity of Tris for the cryptomonad flagellate was removed by Mg + K. However the toxicity of 20 mg. per cent Tris for Volvox globator could not be counteracted by Ca, Mg, Na, K, or trace metals: which implied that Tris might in some organisms interfere also with certain organic metabolites. Indeed the noxious blue-green alga Microcystis aeruginosa is inhibited by 0.01 M Tris at pH 8.4 (Zehnder and Gorham, 1960). At this concentration, they point out, the buffering is negligible. Interference with K utilization might be expected to exaggerate shock states. An exceptional sensitivity of 0. danica to Tris would argue that it * The work reported in this article wasaided by grants from the American Cancer Society, New York, N.Y. by Grant CY-4182(C2) from the National Cancer Institute, Public Health Service, Bethesda, Md., and by grants from the Loomis Institute, New York, N.Y. t Present address: Department of Genetics, Stanford University, Stanford, Calif.

Effect of Sulfonamides on Toxic and Antigenic Actions of Endotoxins of Certain Gram-Negative Bacteria.

Summary Orally administered sulfanilamide, sulfathiazole, sulfapyridine, sulfadiazine, sulfamerazine, and sulfaguanidine confer an equivalent but limited protective effect against the lethal action of intraperi-toneally-administered endotoxin of Salmonella typhimurium. Sulfanilamide has a similar effect on the endotoxin of Shigella paradysenteriæ Flexner. Sulfathiazole administered during the immunization of mice with Salmonella endotoxin appears not to interfere with the immunizing process, as determined by degree of resistance to the lethal action of the endotoxin. Reasons are advanced to support the view that these results apply to other sulfonamide drugs and to the endotoxins of gram-negative bacteria generally.

Action of Bacterial Toxins on Tumors. VI. Protection Against Tumor Hemorrhage Following Heterologous Immunization.

Summary Mice immunized with endotoxin preparations of Shigella paradysenteriœ Flexner, Salmonella typhimurium and Rhodospirillum rubrum were found to have been protected against the induction of hemorrhage in implanted tumors. Protection by immunization was found to be about as effective against the induction of tumor hemorrhage when the heterologous organisms were used as when the homologous were used. This finding supports our earlier hypothesis that a common antigenic toxic component is characteristic of gram-negative bacteria generally.

Action of Bacterial Toxins on Tumors. III. Some Biological Properties of Purified Salmonella typhimurium Endotoxin

Conclusion The acetone, phenol, and various formamide fractions in the extraction of Salmonella typhimurium endotoxin when tested on mice were active in respect to (1) lethality, (2) the induction of hemorrhage in implanted tumors, (3) the production of antibodies which protected mice against the toxic material.

ELECTROLYTE REQUIREMENTS OF PROTISTS AND ARCHEOMETABOLISM

Rubey reasons that the ocean was salty and substantially unchanged throughout geological time and that the increment of salts deposited by leaching of the land was counterbalanced by an increase in the amount of water.’ Consequently, if we assume that life began in the sea, electrolytes were very much a part of the environment for primordial life. The occurrence of inorganic electrolytes as essential constituents of organisms might be construed as the indelible imprint of the sea, as Macallum2 regarded the pattern of salts in the body fluids of metazoa. Unfortunately, we are densely ignorant when we consider the question: to which part of the cell are electrolytes indispensable? Metazoan metabolism supplies further incentives for work on the fundamentals of electrolyte requirements. hfovements of Na+ and K+ characterize nervous activity ;3 what mode of intracellular communication preceded this development? To trace the physical basis of thought to its beginnings obviously requires a stupendous knowledge of the nature of life; less ambitiously, it demands an understanding of the mode of action of hormones. Certain steroid hormones drastically influence electrolyte relations in vertebrates; where‘are the protistan forerunners-the Anlugen-of these systems? Our bias, directed by drudgery in the pursuit of indispensable trace elements and growth factors that lie well beyond the limits of sensitivity of conventional chemical analysis, makes us wonder at the current preoccupation with those quantitatively most abundant linear macromolecules, the nucleic acids and fibrous proteins that are invariant cell constituents, and with such conspicuous molecules as adenosine triphosphate (ATP). Investigation of the role of quantitatively minute invariants can be postponed only a t the peril of impeding an increase in our knowledge of these macroconstituents. We are inclined to look upon certain inorganic electrolytes as indispensable to life and as capable of revealing much about its nature. While nucleic acids and associated protein may well be the repositories of genetic information, how is this information communicated to the effectors of the cell? We find it difficult to conceive of genetic information as merely a punched tape. The means of communication are part of this information, and the problem of communication is inseparable from that of the storage and duplication of information. In short, experiments designed to trace the ancestry of neurohumoral mechanisms, in which electrolytes play an important role, should bring one close to what the French call the “secret of life.” Some components of the original cell machine must have composed the substratum for the evolution of the neurohumoral systems that underlie thought, as closely as any reaction chains can be said to do this. The narrower problem considered here is: Can the electrolyte requirements of extant organisms provide clues to the origin of these systems?

A Cross-Protective Reaction Between Moccasin Venom and the Endotoxin of Salmonella typhimurium.

Summary Mice immunized with moccasin venom were protected against otherwise lethal doses of the endotoxin of Salmonella typhimurium; and, inversely, salmonella-immunized mice were protected against the venom. This cross-protection may be due to the presence in gram-negative organisms and moccasin venom of a common factor characterized by hemorrhagic action, antigenicity, and a lack of serological specificity.